纤连蛋白过表达抑制小梁网细胞单层通透性。

Fibronectin overexpression inhibits trabecular meshwork cell monolayer permeability.

作者信息

Li An-Fei, Tane Nobuhiro, Roy Sayon

机构信息

Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.

出版信息

Mol Vis. 2004 Oct 7;10:750-7.

PMID:15496827

Abstract

PURPOSE

To study whether excess synthesis of an extracellular matrix (ECM) component, fibronectin (FN), underlying the monolayer of human trabecular meshwork (HTM) cells, influences permeability.

METHODS

To upregulate FN expression, HTM cells were grown in high glucose (30 mM) medium for 10 days. In parallel, cells were grown in normal (5 mM) medium as control, and two separate groups of HTM cells were grown in high glucose medium for transfection with FN antisense phosphorothioate oligonucleotides (AS-FN oligos) to modulate high glucose induced FN overexpression, or random phosphorothioate oligonucleotides (Ran oligos) as control. FN protein expression and distribution was assessed by western blot analysis and immunofluorescence microscopy. In parallel, HTM cells were grown in transwell plates in normal or high glucose medium to perform in vitro permeability (IVP) assays and to assess transelectrical resistance (TER).

RESULTS

Western blot analysis showed FN expression was upregulated by 27% (p=0.018) in HTM cells grown in high glucose medium compared to cells grown in normal medium. Immunofluorescence microscopy showed intense FN immunostaining, and IVP results showed a consistent reduction in monolayer permeability (13% reduction, p=0.004) in cells grown in high glucose medium compared to cells grown in normal medium. When cells grown in high glucose medium were transfected with AS-FN oligos FN expression was reduced by 33% (p=0.009) and resulted in increased permeability to near normal levels (98+/-7% of control, p=0.01), whereas random oligos had no effect on either FN expression or IVP. TER was significantly increased across TM cell monolayers grown in high glucose compared to those grown in normal medium (143+/-11% of control, p=0.001), which was reduced when cells were transfected with AS-FN oligos (109+/-7% of control, p=0.02) whereas cells transfected with random oligos showed no change.

CONCLUSIONS

Excess FN synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow associated with the development of primary angle open glaucoma (POAG).

摘要

目的

研究人小梁网（HTM）细胞单层下方细胞外基质（ECM）成分纤连蛋白（FN）的过度合成是否会影响通透性。

方法

为上调FN表达，将HTM细胞在高糖（30 mM）培养基中培养10天。同时，将细胞在正常（5 mM）培养基中培养作为对照，另外两组HTM细胞在高糖培养基中培养，用FN反义硫代磷酸酯寡核苷酸（AS - FN寡核苷酸）转染以调节高糖诱导的FN过表达，或用随机硫代磷酸酯寡核苷酸（Ran寡核苷酸）作为对照。通过蛋白质印迹分析和免疫荧光显微镜评估FN蛋白的表达和分布。同时，将HTM细胞在正常或高糖培养基中的Transwell板中培养，以进行体外通透性（IVP）测定并评估跨膜电阻（TER）。

结果

蛋白质印迹分析显示，与在正常培养基中培养的细胞相比，在高糖培养基中培养的HTM细胞中FN表达上调了27%（p = 0.018）。免疫荧光显微镜显示强烈的FN免疫染色，IVP结果显示，与在正常培养基中培养的细胞相比，在高糖培养基中培养的细胞单层通透性持续降低（降低13%，p = 0.004）。当在高糖培养基中培养的细胞用AS - FN寡核苷酸转染时，FN表达降低了33%（p = 0.009），并导致通透性增加至接近正常水平（为对照的98±7%，p = 0.01），而随机寡核苷酸对FN表达或IVP均无影响。与在正常培养基中培养的细胞相比，在高糖培养基中培养的TM细胞单层的TER显著增加（为对照的143±11%，p = 0.001），当细胞用AS - FN寡核苷酸转染时TER降低（为对照的109±7%，p = 0.02），而用随机寡核苷酸转染的细胞则无变化。